1. Field of the Invention
This invention provides compositions and methods for profiling transcription factor activity. In particular, the invention provides nucleic acid constructs containing protein binding sites and methods for detecting and measuring the binding of proteins and particularly transcription factors to the binding sites in these constructs.
2. Background and Related Art
Eukaryotes are composed of specialized cell types that are organized into tissues and organs. Regardless of cell type and function, all cells within an individual eukaryotic organism contain the same set of genes referred to as the genome. Differences between cells arise through the differential expression of genes. Expression of individual genes is controlled through the binding of proteins to regulatory sequences of DNA in the genome such as promoters and repressors. Protein binding to such control sequences can cause an increase or decrease in the rate of transcription of a gene. These DNA-binding proteins, called transcription factors, regulate gene transcription and thereby control all of the essential characteristics of a cell including cellular reproduction, development and differentiation, response to environmental stimuli, and tissue homeostasis in normal and disease states. Transcription factors comprise hundreds of specialized proteins that regulate gene expression by either facilitating or inhibiting the enzyme RNA polymerase in the initiation and maintenance of transcription.
The activation or inhibition of regulatory transcription factors occurs as a downstream event in signal transduction cascades that are initiated by perturbations such as a change in the oxidation state of a cell, or the binding of a ligand to its cell surface receptor. In the case of cell surface receptors, a ligand-binding event may trigger signaling cascades that fan out to regulate multiple genes that contribute to biological responses. Cross talk between signal transduction systems also is common, with disparate stimuli utilizing many of the same protein kinases, phosphatases and second messenger systems. Highly refined regulation of biological responses, therefore, occurs as webs of interacting signaling systems involving kinases, phosphatases and second messengers triggered by each stimulus that culminate in qualitatively and/or quantitatively different sets of transcription factor activation. It has been estimated that there are approximately 1000 transcription factors in the human genome that contribute the specificity to regulate the independent expression of approximately 40,000 genes. Consequently, biological responses that are characterized by changes in gene expression may be defined by distinct signatures of transcription factor activation. Transcription factors, therefore, are of significant interest as targets to affect specific individual or global changes in gene expression.
Cell surface receptors have been a primary focus of pharmaceutical research and comprise the majority of therapeutic targets. These receptor-targeted strategies have been successful in treating disease and prolonging life, but most of these therapies suffer from a lack of specificity. In the majority of cases, cell surface receptors are multifunctional. For instance, a given receptor may reside on different cell types, and activate intricate webs of signaling cascades to regulate multiple biological responses. A well-characterized example of the multi-functional nature of receptors is the insulin receptor. Insulin receptors are broadly distributed in diverse tissues and activate multiple second messenger systems to directly affect metabolic responses ranging from glucose homeostasis to lipolysis, platelet aggregation, and more recently, the formation of memory (1–3).
The consequence of a multifunctional role for individual receptors is that many drugs on the market today have detrimental side effects that exact an enormous cost on society and the pharmaceutical industry. The need to better define biological responses to potential therapeutic agents and to more fully understand the nature of potential therapeutic targets has spurred great interest in the development and application of DNA microarrays for comparative gene analysis.
The result of this interest has been many advancements and successes using gene chip technology. By screening tens of thousands of genes on DNA microarrays, patterns or profiles of gene expression comprising up to several hundred genes have been used to diagnose and classify specific diseases (5). Nonetheless, obtaining profiles of gene expression that increase understanding of disease processes has proven difficult in many cases due to the complexity of the diseases.
In these circumstances, profiling transcription factor activity may provide an alternative means to diagnose and classify disease. In contrast to results obtained from gene expression profiling of mRNA, reports have demonstrated that significant qualitative and quantitative differences in transcription factor activation are associated with and may control the expression of disease-associated genes responsible for the onset and progression of infectious diseases, autoimmune, inflammatory, neurological, circulatory (14) and cardiovascular diseases (15,17), obesity (18) and cancer (15,16,19). Transcription factors have been demonstrated to have diagnostic (5) and prognostic (6) applications and have been identified as targets for therapeutic intervention into cancer and inflammatory diseases (4). Unfortunately, progress in transcription factor targeted therapy and transcription-factor based diagnostic and prognostic application has been slow due to the bottleneck that exists for screening large numbers of samples for multiple transcription factors. Currently, no technology is available for the rapid comprehensive profiling of the activity of multiple transcription factors.
Conventional methods of detecting and measuring DNA-binding proteins such as transcription factors include the electrophoretic mobility shift assay (EMSA) (24), supershift EMSA (25), and ELISA-based techniques. The EMSA or gel-shift assay provides a simple and rapid method for detecting DNA-binding proteins such as transcription factors, and has been widely used. The assay is based on the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The EMSA is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or whole cell extract preparations), with a labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed by electrophoresis on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by performing competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences. Gel-shift assays typically use radioactively-labeled DNA probes, but non-radioactive labels such as biotin or fluorescent dyes can also be used. This method is not suited, however, for rapid screening of large numbers of samples or multiple transcription factors simultaneously.
The supershift-EMSA is a complement to the gel shift assay that allows specific identification of the DNA-bound protein using specific antibodies. The supershift-EMSA is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or whole cell extract preparations), with a labeled or unlabeled DNA fragment containing the putative protein binding site and an antibody to the putative protein. The reaction products are then analyzed by electrophoresis on a non-denaturing polyacrylamide gel and the DNA-protein-antibody complex can be detected by detecting the label on the DNA or by using an antibody to detect the antibody in the DNA-protein-antibody complex. Again, the specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences. The “super-complex” of DNA-protein-antibody has significantly reduced mobility than the DNA-protein complex when subjected to electrophoresis in non-denaturing gels. Although useful for basic research, gel-shift and supershift assays have low sensitivity and very low throughput due to the large amount of handling that must be performed. Furthermore, the gel-shift and supershift assays are not quantitative and can only detect the presence or absence of a particular DNA-binding protein.
Recently, ELISA techniques have become available for detection of known DNA-binding proteins (22). In these ELISA assays, DNA fragments containing a putative protein binding site are bound to a solid phase such as the bottoms of the wells of a 96-well polystyrene plate. The sample containing a purified protein, or a complex mixture of proteins (such as nuclear or whole cell extract preparations) is incubated in the well containing the immobilized DNA fragment containing the putative protein binding site. The well is then washed to remove all non-bound components of the sample, and an antibody specific for the putative bound protein is added. Binding of the antibody is accomplished using standard ELISA techniques with colorimetric, fluorescent, or chemiluminescent detection.
ELISA assays are roughly 10-fold more sensitive than gel-shift assays and can be adapted to high-throughput analysis. However, they suffer a major disadvantage in that the target protein binding sequences must be known, and antibodies must be available to detect the bound protein. Thus, they are limited to studying systems that have already been well-characterized. Furthermore, these assays cannot be multiplexed and, accordingly, the sample volume required to obtain a panel of DNA-binding markers precludes the broad use of this technique for generating DNA-protein binding profiles.
A multiplex transcription factor assay based on a combination of gel shift and DNA chip technology has also been recently described (23). In this assay a nuclear extract is incubated with a pool of biotin-labeled double-stranded oligonucleotides. The protein-bound oligonucleotides are electrophoresed, and the portion that have gel-shifted are excised from the gel and eluted. The sequences of the oligonucleotides are then determined by hybridization to a membrane array. Although this technique is multiplexed and can provide a transcription factor profile, it involves multiple steps and requires many manipulations that must be performed by hand it and therefore is unsuitable for moderate or high-throughput analysis.
It is apparent, therefore, that compositions and methods that permit simultaneous detection of multiple DNA-binding proteins in a multiplex or array format, and that provide profiles of DNA binding activity by proteins, specifically, transcription factors, are greatly to be desired. In particular, it is highly desirable to develop assays that allow detection and measurement of multiple protein-DNA binding events in a single sample.
The present invention therefore provides novel compositions and assay methods that permit specific detection of DNA-binding proteins. In particular, the present invention represents a substantial improvement over the prior art in that it provides a quantitative output without the need for specific antibodies or protein binding reagents. Furthermore, the present invention does not result in the release of a soluble signaling molecule so that detection of DNA-binding proteins can be performed in a solid- or liquid-array format, thereby facilitating the use of signal amplification techniques that cannot be used when a soluble signal is generated.